日本七鳃鳗(Lampetra japonica)口腔腺表达序列标签(EST)分析

以日本七鳃鳗口腔腺为材料,构建库容量为2.1×106pfu/mL的cDNA文库。通过对文库中克隆子的序列测定和生物信息学初步分析,得到1323条有效EST序列。经BlastX及BlastN软件进行同源对比分析,653条(49.36%)EST可在蛋白质或核苷酸水平上找到同源序列,其中328条与七鳃鳗科物种同源。同源序列功能分类大致分为11类,与蛋白质合成有关的蛋白所占比例最大。1323条EST进行片段重叠群分析(contig analysis)获得包括547条序列在内的162组片段重叠群并确定了8条全长cDNA。日本七鳃鳗口腔腺cDNA文库以及EST文库的成功构建,为研究日本七鳃鳗口腔腺的功能基因和蛋白质组学奠定了基础。     

Gateway技术构建菊芋块茎全长cDNA文库及EST测序分析

为丰富菊芋功能基因组学研究基础平台,以成熟期菊芋块茎为材料,将菊芋全长cDNA与Gateway供体载体pDONR222重组,构建了菊芋非剪切型全长cDNA文库。文库质量分析表明:未经扩增的原始文库库容量为5.76×10~6 CFU,插入片段大小主要为1～3000 bp,重组率为100%(24/24),达到了高质量文库的标准。利用该文库进行表达序列标签(expressed sequence tag, EST)测序,得到2639条高质量的EST序列,拼接后获得1895条非重复的唯一表达序列(unigene)。与NCBI的NR数据库同源比对分析表明,共有1533条unigene(80.9%)与已知基因有显著的同源性。GO分类结果显示:菊芋块茎表达基因在分子功能类群中,结合和催化活性所占比例最高。此cDNA文库将可用于菊芋功能基因组研究、新基因筛选、高通量EST测序以及菊芋cDNA芯片的制备等。     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapidamplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapid amplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

猪电压依赖性阴离子通道1基因的分离及物理定位

从猪胚胎骨骼肌cDNA文库中筛选出一克隆子,通过测序及电子延伸获得包含全长CDS的猪VDAC1基因cDNA序列。比对发现此基因在核苷酸和氨基酸水平与人及小鼠都具有较高的同源性。应用辐射杂种板(RH)对此基因进行染色体的精确定位,定位结果显示VDAC1基因定位在猪2号染色体长臂。     

一种新的人端粒相关锌指蛋白cDNA的克隆及鉴定

为了从早期胚胎寻找与发育分化有关的新基因,本文构建了3周龄人胚cDNA文库,并应用EST技术对该文库中随机挑选的47个低丰度克隆进行测序,结果发现了一个与人亚端粒DNA和锌指基因同源的cDNA克隆(L30),该基因长约3.8kb,5＇端序列有明显的阅读框架(ORF),3＇端序列有加尾信号(AAUAGA)和有39个A组成的Poly(A)尾巴;通过Northern杂交确认在早期人胚胎中有表达,应用地高辛染色体原位杂交技术将其定位于人第12号染色体长臂端部.     

火炬松热胁迫cDNA文库的表达谱分析

以火炬松热胁迫cDNA文库的EST序列为材料,对EST序列进行聚类、拼接等处理后,再进行Blast同源比对以及基因GO注释分析。研究结果如下:从Forest TreeDB数据库中下载了火炬松热胁迫cDNA文库的所有EST序列,共4 283条。EST序列经CAP3拼接后,获得2 062个UniGene,其中934个Contig,1 128个Singletons。对UniGene进行同源检索,按照GO的分子功能、生物过程和细胞组分三个不同分类角度分类,被赋予功能的基因数累计达4 661个,但365个(17.7%)的序列与核酸和蛋白数据库无序列同源性,即17.7%为新发现的基因。经对所有具有功能的基因研究发现,受外界胁迫表达的抗逆相关基因含量较高。上述研究结果对于研究火炬松热胁迫基因表达特征与抗逆分子机制具有一定的借鉴价值,以及开发火炬松新分子标记与开展分子辅助育种具有一定的指导意义。     

人类β-1,4-半乳糖苷转移酶Ⅲ基因的分离与克隆

以人、牛、鼠、鸡、蜗牛的β-1,4-半乳糖苷转移酶催化区的编码核苷酸序列为探针,在NCBI GenBank EST数据库中进行同源搜寻,获得若干有高度同源的EST.在拼接序列的两端设计引物,以从人胎盘cDNA文库中PCR扩增获得的片段为探针,在人胎盘cDNA分子库中步移获得一个长1,907bp的cDNA片段,包含一个长1,179bp的开放阅读框(ORF,open reading frame),编码393个氨基酸残基。该基因与已知的人类β1,4-GalTI的氨基酸同源性为43.8%,在蛋白质催化区的同源性更高达60.9%。表达谱分析发现该基因在人体16种组织中均有不同程度的表达,转录本大小约为2.4kb.通过该cDNA人/啮齿类杂种细胞株DNA Southern杂交将该基因定位在1号染色体。     

基于PC/Linux的核酸序列电子延伸系统的构建及其应用

新基因全长cDNA序列的获得常常是分子生物学工作者面临的难题。人类基因组计划及其相关计划的实施导致了大量表达序列标签(EST)的产生。利用一定的生物信息学算法,这些EST序列往往可用来对新基因片段进行延伸。采用Linux操作系统,利用Blast软件和Phrap软件以及EST数据库在微机上构建了EST序列的电子延伸系统,并对来自于人胎肝的11386条EST序列和511条插入片段全长cDNA序列进行了电子延伸,结果显示8373条EST序列和389条插入片段全长cDNA序列得到了程度不等的延伸,部分结果通过RACE实验得到证实。该套系统可高效地、规模化进行EST序列的延伸,可为通过实验获得新基因全长cDNA序列提供重要线索。 Abstract:Normally it is difficult to obtain full-length cDNA sequence of novel genes.More and more expressed sequence tags(ESTs) have been obtained since the start-up of human genome project.Powerful system is badly needed for data mining on these EST sequences.Based on a personal computer coupled with Linux operating system and EST database,the Blast software and Phrap software were used to construct a platform for in silico elongation of ESTs in our lab.The performance was tested using 11386 EST sequences and 511 partial-length cDNA sequences.Results demonstrated that 8373 EST and 389 cDNA sequence were elongated using this system.Thus the platform seems to be a fast way for full-length cDNA sequence cloning of new genes.     

EST clustering error evaluation and correction

MOTIVATION: The gene expression intensity information conveyed by (EST) Expressed Sequence Tag data can be used to infer important cDNA library properties, such as gene number and expression patterns. However, EST clustering errors, which often lead to greatly inflated estimates of obtained unique genes, have become a major obstacle in the analyses. The EST clustering error structure, the relationship between clustering error and clustering criteria, and possible error correction methods need to be systematically investigated. RESULTS: We identify and quantify two types of EST clustering error, namely, Type I and II in EST clustering using CAP3 assembling program. A Type I error occurs when ESTs from the same gene do not form a cluster whereas a Type II error occurs when ESTs from distinct genes are falsely clustered together. While the Type II error rate is <1.5% for both 5' and 3' EST clustering, the Type I error in the 5' EST case is approximately 10 times higher than the 3' EST case (30% versus 3%). An over-stringent identity rule, e.g., P >/= 95%, may even inflate the Type I error in both cases. We demonstrate that approximately 80% of the Type I error is due to insufficient overlap among sibling ESTs (ISO error) in 5' EST clustering. A novel statistical approach is proposed to correct ISO error to provide more accurate estimates of the true gene cluster profile.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

Eighteen Novel Human Genes Regionally Mapped on Chromosome 11

Expression sequence tags (EST) obtained by sequencing a randomlyprimed cDNA library and gene signatures (GS) obtained by sequencinga 3'-directed cDNA library can identify genes that are activein the source cells. Eight ESTs and ten GSs which representnovel human genes, except for one GS, and which have been assignedto human chromosome 11 were used to select cosmids from a chromosome11-specific cosmid library. These cosmids were regionally mappedusing the fluorescence in situ hybridization technique.     

Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is the most important fungal disease of rice. To understand the molecular basis of interaction between the fungus and rice, we constructed a cDNA library from a rice-resistant line inoculated with M. oryzae. One hundred and fifty-three cDNA clones were sequence analyzed, of which 129 exhibited significant nucleotide sequence homology to known genes, 21 were homologous to unknown genes, while three clones did not match to any database. However, these three unmatched clones showed sequence homology at protein level in the protein databases and one of them encoded a disease resistance-related protein kinase and was abundant in the EST collection. Northern analysis showed that this disease resistance-related protein kinase gene was induced by inoculation and only expressed in the rice-resistant, but not susceptible, lines. Southern analysis showed that this gene was present in a single copy in the rice genome and co-segregated with the M. oryzae resistance in the cross of the resistant and susceptible lines. This study illustrates that sequencing of ESTs from inoculated resistant plants can reveal genes responsive to pathogen infection, which could help understand plant defense mechanisms.     

ESTminer: a suite of programs for gene and allele identification

SUMMARY: ESTminer is a collection of programs that use expressed sequence tag (EST) data from inbred genomes to identify unique genes within gene families. The algorithm utilizes Cap3 to perform an initial clustering of related EST sequences to produce a consensus sequence of a gene family. These consensus sequences are then used to collect all ESTs in the original EST library that are related using BLAST. A redundancy based criterion is applied to each EST to identify reliable unique gene-sequences. Using a highly inbred genome as a source of ESTs eliminates the necessity of computing covariance on each polymorphism to identify alleles of the same gene, thus making this algorithm more streamlined than other alternatives which must computationally attempt to distinguish genes from alleles. AVAILABILITY: The programs were written in PERL and are freely available at http://www.soybase.org/publication_data/Nelson/ESTminer/ESTminer.html CONTACT: nelsonrt@iastate.edu SUPPLEMENTARY INFORMATION: Figures and dataset can be obtained from: http://www.soybase.org/publication_data/Nelson/ESTminer/ESTminer.html.     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.